SB225002 Induces Cell Death and Cell Cycle Arrest in Acute Lymphoblastic Leukemia Cells through the Activation of GLIPR1.

Acute Lymphoblastic Leukemia (ALL) is the most frequent childhood malignancy. In the effort to find new anti-leukemic agents, we evaluated the small drug SB225002 (N-(2-hydroxy-4-nitrophenyl)-N'-(2-bromophenyl)urea). Although initially described as a selective antagonist of CXCR2, later studies have identified other cellular targets for SB225002, with potential medicinal use in cancer. We found that SB225002 has a significant pro-apoptotic effect against both B- and T-ALL cell lines. Cell cycle analysis demonstrated that treatment with SB225002 induces G2-M cell cycle arrest. Transcriptional profiling revealed that SB225002-mediated apoptosis triggered a transcriptional program typical of tubulin binding agents. Network analysis revealed the activation of genes linked to the JUN and p53 pathways and inhibition of genes linked to the TNF pathway. Early cellular effects activated by SB225002 included the up-regulation of GLIPR1, a p53-target gene shown to have pro-apoptotic activities in prostate and bladder cancer. Silencing of GLIPR1 in B- and T-ALL cell lines resulted in increased resistance to SB225002. Although SB225002 promoted ROS increase in ALL cells, antioxidant N-Acetyl Cysteine pre-treatment only modestly attenuated cell death, implying that the pro-apoptotic effects of SB225002 are not exclusively mediated by ROS. Moreover, GLIPR1 silencing resulted in increased ROS levels both in untreated and SB225002-treated cells. In conclusion, SB225002 induces cell cycle arrest and apoptosis in different B- and T-ALL cell lines. Inhibition of tubulin function with concurrent activation of the p53 pathway, in particular, its downstream target GLIPR1, seems to underlie the anti-leukemic effect of SB225002.

N-(1'-naphthyl)-3,4,5-trimethoxybenzohydrazide as microtubule destabilizer: Synthesis, cytotoxicity, inhibition of cell migration and in vivo activity against acute lymphoblastic leukemia.

Tubulin-interacting agents, like vinca alkaloid and taxanes, play a fundamental role in cancer chemotherapy, making cellular microtubules (MT), one of the few validated anticancer targets. Cancer resistance to classical MT inhibitors has motivated the development of novel molecules with increased efficacy and lower toxicity. Aiming at designing structurally-simple inhibitors of MT assembly, we synthesized a series of thirty-one 3,4,5-trimethoxy-hydrazones and twenty-five derivatives or analogs. Docking simulations suggested that a representative N-acylhydrazone could adopt an appropriate stereochemistry inside the colchicine-binding domain of tubulin. Several of these compounds showed anti-leukemia effects in the nanomolar concentration range. Interference with MT polymerization was validated by the compounds' ability to inhibit MT assembly at the biochemical and cellular level. Selective toxicity investigations done with the most potent compound, a 3,4,5-trimethoxy-hydrazone with a 1-naphthyl group, showed remarkably selective toxicity against leukemia cells in comparison with stimulated normal lymphocytes, and no acute toxicity in vivo. Finally, this molecule was as active as vincristine in a murine model of human acute lymphoblastic leukemia at a weekly dose of 1 mg/kg.

The role of CXCR2 chemokine receptors in the oral squamous cell carcinoma.

This study evaluated the relevance of CXCR2 chemokine receptors in oral squamous cell carcinoma, by means of in vitro and in vivo approaches. The in vitro incubation of the selective and non-peptide CXCR2 receptor antagonist N-(2-hydroxy-4-nitrophenyl)-N9-(2-bromophenyl) Urea (SB225002; 25 to 800 nM) produced a time- and concentration-dependent inhibition of SCC158 (rat) and HN30 (human) cell lines viability. Conversely, this antagonist did not significantly affect the viability of the immortalized keratinocyte lineage, HaCaT. Additionally, the incubation of human IL-8 and rat CINC-1 CXCR2 agonists produced a concentration-related increase on HN30 and SCC158 proliferation. The submucosal injection of SCC158 cells (5 × 10(6) cells) into the tongue of Fischer 344 rats induced tumor development, which displayed typical clinical features. Immunohistochemical analysis of rat tongue biopsies revealed a marked increase of CXCR2 receptor immunoreactivity, which was accompanied by augumented expression of VEGF and caspase-3. Our data suggests an important role for CXCR2 receptors in oral squamous cell carcinoma.

Antinociceptive Activity of Trichilia catigua Hydroalcoholic Extract: New Evidence on Its Dopaminergic Effects.

Trichilia catigua is a native plant of Brazil; its barks are used by some local pharmaceutical companies to prepare tonic drinks, such as Catuama. The present study was addressed to evaluate the effects of T. catigua hydroalcoholic extract in mouse nociception behavioral models, and to evaluate the possible mechanisms involved in its actions. Male Swiss mice were submitted to hot-plate, writhing and von Frey tests, after oral treatment with T. catigua extract (200 mg kg(-1), p.o.). The extract displayed antinociceptive effect in all three models. For characterization of the mechanisms involved in the antinociceptive action of the extract, the following pharmacological treatments were done: naloxone (2.5 mg kg(-1), s.c.), SR141716A (10 mg kg(-1), i.p.), SCH23390 (15 µg kg(-1), i.p.), sulpiride (50 mg kg(-1), i.p.), prazosin (1 mg kg(-1), i.p.), bicuculline (1 mg kg(-1), i.p.) or dl-p-chlorophenylalanine methyl ester (PCPA, 100 mg kg(-1), i.p.). In these experiments, the action of T. catigua extract was evaluated in the hot-plate test. The treatment with SCH23390 completely prevented the antinociceptive effect, while naloxone partially prevented it. The possible involvement of the dopaminergic system in the actions of T. catigua extract was substantiated by data showing the potentiation of apomorphine-induced hypothermia and by the prevention of haloperidol-induced catalepsy. In conclusion, the antinociceptive effects of T. catigua extract seem to be mainly associated with the activation of dopaminergic system and, to a lesser extent, through interaction with opioid pathway.

Effects of the compounds MV8608 and MV8612 obtained from Mandevilla velutina in the model of hemorrhagic cystitis induced by cyclophosphamide in rats.

Hemorrhagic cystitis (HC) is a common side effect observed in patients under chemotherapy with cyclophosphamide (CYP). The urotoxic side effects of CYP are attributed to the metabolic compound acrolein, and can be partially prevented by the uroprotector agent 2-mercaptoethene sulfate (Mesna). The present study analyzed the anti-inflammatory and the antinociceptive effects of compounds MV8608 and MV8612 obtained from Mandevilla velutina in the rat model of CYP-induced HC. Male Wistar rats were used (six to eight per group, 220-250 g). HC was induced by a single administration of CYP (100 mg/kg, ip). Three behavioral parameters--breathing rate, closing of the eyes, and specific posture--were used as nociception indexes, and scored at different time intervals (15-180 min) after cystitis induction. As inflammatory parameters, hemorrhage presence, edema formation, and bladder weight were determined at 24 h after CYP administration. The neutrophil migration was assessed by means of myeloperoxidase (MPO activity), 4 h after cystitis induction. As expected, Mesna treatment was able to reduce in a significant manner all the inflammatory and the nociceptive parameters induced by CYP. Of note, the administration of MV8608 significantly inhibited the hemorrhage formation and the neutrophil recruitment, while the MV8612 treatment markedly reduced the bladder weight, without interfering with neutrophil influx. Interestingly, the treatment with either MV8608 or MV8612 markedly reduced the nociceptive responses. The present results clearly indicate that MV8608 and MV8612 might represent important alternatives to prevent side effects, especially the nociception, following chemotherapy with CYP.

Involvement of phosphoinositide 3-kinase gamma in the neuro-inflammatory response and cognitive impairments induced by beta-amyloid 1-40 peptide in mice.

Alzheimer disease (AD) is the most common form of dementia in the elderly, and the neuro-pathological hallmarks of AD include neurofibrillary tangles (NFT), and deposition of beta-amyloid (Abeta) in extracellular plaques. In addition, chronic inflammation due to recruitment of activated glial cells to amyloid plaques are an invariant component in AD, and several studies have reported that the use of non-steroidal anti-inflammatory drugs (NSAIDs) may provide a measure of protection against AD. In this report we have investigated whether phosphoinositide 3-kinase gamma (PI3Kgamma), which is important in inflammatory cell migration, plays a critical role in the neuro-inflammation, synaptic dysfunction, and cognitive deficits induced by intracerebroventricular injection of Abeta(1-40) in mice. We found that the selective inhibitor of PI3Kgamma, AS605240, was able to attenuate the Abeta(1-40)-induced accumulation of activated astrocytes and microglia in the hippocampus, and decrease immuno-staining for p-Akt and cyclooxygenase-2 (COX-2). Interestingly, Abeta(1-40) activated macrophages treated with AS605240 or another PI3Kgamma inhibitor, AS252424, displayed impaired chemotaxis in vitro, but their expression of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) was unaffected. Finally, AS605240 prevented Abeta(1-40)-induced cognitive deficits and synaptic dysfunction, but failed to modify scopolamine-induced amnesia. Our data suggests that inhibition of PI3Kgamma may represent a novel therapeutic target for treating AD patients.

The effects of the selective and non-peptide CXCR2 receptor antagonist SB225002 on acute and long-lasting models of nociception in mice.

This study evaluated the antinociceptive effects of the selective and non-peptide CXCR2 antagonist SB225002 in mouse models of pain. As assessed in different tests of spontaneous nociception, intraperitoneal (i.p.) administration of SB225002 caused consistent and dose-related reduction of acetic acid-induced abdominal constrictions, whereas it did not significantly affect the nociception evoked by formalin, capsaicin, glutamate or phorbol ester acetate (PMA). Systemic treatment with SB225002 strikingly reduced the spontaneous nociception induced by 8-bromo-cAMP (8-Br-cAMP), or mechanical hypernociception induced by prostaglandin E(2) (PGE(2)), epinephrine, or the keratinocyte-derived chemokine (KC). In the carrageenan model, SB225002 markedly reduced mechanical hypernociception when administered by i.p., intrathecal (i.t.) or intracerebroventricular (i.c.v.) routes, or even when co-administered with carrageenan into the mouse paw, indicating peripheral and central sites of action for SB225002. In addition, i.p. treatment with SB225002 significantly attenuated the increase in MPO activity or the elevation of IL-1beta, TNFalpha or KC levels following carrageenan injection. In the persistent models of pain evoked by complete Freund's adjuvant (CFA) or by the partial ligation of the sciatic nerve (PLSN), the repeated administration of SB225002 displayed prominent and long-lasting antinociceptive effects. Notably, SB225002 did not evoke unspecific central effects, as evaluated in the open-field and rota-rod tests, or even in the latency responses for thermal stimuli. Our data confirm the previous notion on the critical role exerted by chemokines in pain, indicating that selective CXCR2 antagonists, such as SB225002, might well represent interesting and innovative alternatives for the management of both acute and chronic pain.

Gallic acid ester derivatives induce apoptosis and cell adhesion inhibition in melanoma cells: The relationship between free radical generation, glutathione depletion and cell death.

Malignant melanoma is a lethal disease, and the incidence and mortality associated with it are increasing worldwide. It has a significant tendency to develop both metastasis and resistance to chemotherapy. The tumor cells show abnormal redox regulation, and although the molecular mechanisms involved are not well characterized, they seem to be related to oxidative stress. In a previous study, we showed the antitumoral properties of gallic acid ester derivatives in leukemia cells. Here, we show the effect of octyl, decyl, dodecyl and tetradecyl gallates on B16F10 cells, a melanoma cell line. All compounds induced cytotoxic effects, and the IC(50) values obtained were between 7microM and 17microM after 48h of incubation. Cell death occurred through apoptosis, as demonstrated by the genomic DNA fragmentation pattern. The gallates were able to induce significant production of free radicals, deplete both glutathione and ATP, activate NF-kappaB and promote the inhibition of cell adhesion under the experimental conditions. The glutathione depletion induced by these compounds was related to the inhibition of gamma-glutamylcysteine synthase activity. These results suggest that gallates induce tumoral cell death through apoptosis as a consequence of oxidative stress, though they use different mechanisms to do so. These findings are important since melanoma cells are resistant to death because of their high level of antioxidant defense, adhesion capability and propensity to metastasize.

Non-peptide antagonists for kinin B1 receptors: new insights into their therapeutic potential for the management of inflammation and pain.

Kinin B1 and B2 receptors are central to the aetiology of pain and inflammation. Constitutive B2 receptors are commonly associated with the acute phase of inflammation and nociception, whereas the inducible B1 receptors are mostly linked to the chronic or persistent phase (or both). Therefore, selective, orally active kinin B1 receptor antagonists could be potentially therapeutic. B1 receptor antagonists have long been exclusively peptides, but recently a few non-peptide representatives have been identified. The clinical potential of these non-peptide molecules has not yet been evaluated, but they might have a role in treating persistent inflammation and pain, especially when no satisfactory therapy is available. This review summarizes recent advances in the identification and the potential therapeutic properties of these molecules.

Relationship between the lipophilicity of gallic acid n-alquil esters' derivatives and both myeloperoxidase activity and HOCl scavenging.

The gallic acid and several n-alkyl gallates, with the same number of hydroxyl substituents, varying only in the side carbonic chain length, with respective lipophilicity defined through the C log P, were studied. It evidenced the structure-activity relationship of the myeloperoxidase activity inhibition and the hypochlorous acid scavenger property, as well as its low toxicity in rat hepatic tissue. The gallates with C log P below 3.0 (compounds 2-7) were more active against the enzyme activity, what means that the addition of 1-6 carbons (C log P between 0.92 and 2.92) at the side chain increased approximately 50% the gallic acid effect. However, a relationship between the HOCl scavenging capability and the lipophilicity was not observed. With these results it is possible to suggest that the gallates protect the HOCl targets through two mechanisms: inhibiting its production by the enzyme and scavenging the reactive specie.

Evaluation of antiviral activity of phenolic compounds and derivatives against rabies virus.

Human rabies is a viral disease with a great impact on public health, mainly on account of its fatal course in the majority of cases. Despite the well-established prophylaxis by immunization, rabies is believed to be responsible for 40,000-70,000 human deaths per year, mostly in endemic areas. Palliative support and experimental protocols to avoid death have been employed with no expressive results, with the exception of a recent human case of recovery from rabies. No antiviral drugs are currently available to fight against this infection. In combination with the prophylaxis, an antiviral drug would be useful for human rabies treatment, providing enhanced protection against the encephalitis caused by the virus. Phenolic compounds are derived from the secondary plant metabolism, although they can also be obtained by synthetic processes. Many studies have shown a great range of pharmacological effects for these substances, including vasodilatation, antiallergenic, antiinflammatory and antiviral properties, among others. In this study, the potential in-vitro anti-rabies activity of 24 synthetic phenolic compounds was evaluated using McCoy cells and PV rabies strain. The cytotoxicity (CC50) was assayed by the MTT method and the antiviral activity (IC50) was estimated by the inhibition of viral cytopathic effects. Isoprinosine and ketamine were used as positive controls. The tested compounds showed selectivity indices (SI=CC50/IC50) ranging from 1.0 to 3.9. Six phenolic compounds failed to inhibit the cytopathic effect to any degree, and four showed SI > or = 3.0. According to these results, some probable structure-activity relationships are suggested. It was observed that the presence of free hydroxyl and ether groups influenced the anti-rabies activity. However, additional studies are required to establish these relationships.

Antifungal activity and studies on mode of action of novel xanthoxyline-derived chalcones.

Chalcones and chalcone-like compounds, most of them new ones, prepared by base-catalyzed condensation of appropriate aldehydes and xanthoxyline, were tested for antifungal properties against a panel of yeasts, hialohyphomycetes as well as dermatophytes with the agar dilution assay. Results indicate that neither the sole presence of a "xanthoxyline-like" substitution pattern nor a 2'-OH substituent on ring A are sufficient for these compounds to have antifungal properties. The chalcone 3-(2-chlorophenyl)-1-(2'-hydroxy-4',6'-dimethoxyphenyl)prop-2-en-1-one, with a Cl atom in the ortho position of benzene ring B showed the best antifungal activity against standardized strains of Trichophyton rubrum (MIC = 12.5 microg/mL) and inhibited all of the ten clinical isolates of T. rubrum tested (MIC at which 50 % [MIC(50)] and 90 % [MIC(90)] of the isolates were inhibited = 12.5 and 25 microg/mL). Regarding its mode of action, the Neurospora crassa assay showed a blotchy appearance in the inhibition halo produced by this chalcone, strongly suggesting that it could act by inhibiting the fungal cell wall. This chalcone seems to be an hyphal malformation inducer, since a clear curling of the hypha was observed in this hazy zone at a magnification of x 400. This work strongly contributes to the knowledge of the antifungal properties of hydroxy-chalcones.

Evaluation of anti-herpetic and antioxidant activities, and cytotoxic and genotoxic effects of synthetic alkyl-esters of gallic acid.

The n-alkyl esters of gallic acid (CAS 13857-8) have a diverse range of uses as antioxidants in food, cosmetics and pharmaceutical industries. Pharmaceutical studies performed with these compounds have found that they have many therapeutic potentialities including anti-cancer, antiviral and antimicrobial properties. However, more interest has been devoted to their antioxidant activity due to the ability to scavenge and reduce reactive oxygen species (ROS) formation. In this study, gallic acid and 14 different alkyl gallates were tested. The cytotoxicity and anti-herpetic (HSV-1, KOS and 29-R strains) activity were studied by using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) colorimetric assay and the cell viability by using the Trypan blue dye exclusion method. The genotoxicity was studied by the Comet assay and the antioxidant activity by using the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging and microsomal lipid peroxidation-inhibiting activities. The results showed that all the tested compounds have anti-herpetic activity at non cytotoxic concentrations with selectivity indices (SI = CC50/EC50) varying from 0.89 to 18.34, depending on the used HSV-1 strain. It was observed that all tested alkyl gallates showed some degree of genotoxicity, at the tested concentrations, except cetyl gallate, at 256.60 micromol/L (p <0.05, t-Student test), probably induced by ROS released by infected cells and/or by the alkyl gallates that were not antioxidants, at the tested concentrations, in which they demonstrated anti-herpetic activity. The hydroxyl groups can induce DNA damage due interactions with some metal ions, which are naturally present in the culture medium supplemented with fetal bovine serum, probably explaining the genotoxicity detected. However, the obtained results showed considerable antioxidant activity at smaller concentrations, when compared to quercetin which is considered as a reference drug due to its already described antioxidant potential: DPPH radical scavenging activity with IC50 values varying from 17 to 31 micromol/L; and microsomal lipid peroxidation-inhibiting activity with IC50 values varying from 21 to 59 micromol/L. It was observed that the presence of hydroxyl groups in these molecules is important for their pharmacological profile, but the length of the lateral carbonic chain does not have considerable influence.